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Starbucks: Mobile Marketing Report (Assessment)

Table of Contents Introduction Discussion Concluding Points Works Cited Introduction It is worth noting that, for several years, Starbucks has been one of the leaders in the industry in terms of mobile marketing and commerce. The company managed to find a successful and effective tool to attract new customers and retain their loyal audience, which brought them not only income but also traffic to the coffee shops. The purpose of this paper is to discuss what other companies can emulate from Starbucks and specify what it can do better in regards to the current mobile marketing efforts. Discussion Importantly, the company’s mobile marketing centers on the loyalty program, which ensures customers get various bonuses and benefits (Bidgoli 189). When clients spend money in coffee shops, they get stars that they can later use for receiving free goods. Customers, who use the app, also get a free drink for their birthday. When users collect enough stars during the game offered by Starbucks, they can unlock new levels with free goods, bonus programs, personalized offers and so on. Therefore, the mobile app not only provides clients with benefits but also serves as an entertainment tool, which pushes them to visit coffee shops more often and boosts loyalty (Bidgoli 190). Therefore, the first aspect that other companies can emulate is building customer loyalty through rewarding user experience. Apart from that, enterprises should consider introducing a built-in payment system similar to the one that Starbucks offers its clients. In particular, in the US, more than 23 million people used the system last year, which meant significantly greater incomes for the company (Richter). Therefore, organizations should consider Square payments, which is the method currently employed by Starbucks, to offer their customers a new client-side solution. Moreover, another feature other companies can emulate is augmented reality. Currently, customers of Starbucks can engage in a coffee-making process or try some other tricks using the app while waiting for their order. This is a great source of entertainment for clients, which brings customer care at coffee shops to a new level (Bidgoli 185). Also, other strengths of the company’s mobile strategy are quick searching for coffee shops nearby based on the user’s location, ordering through an application, and choosing a suitable shop to pick the order. The app also offers quick access to information regarding the duration of order execution. In addition, the loyalty program provides users with the possibility to check the balance of the bonus account in real time. These features are easier to implement rather than virtual reality or payment system; therefore, companies could consider emulating these mobile marketing strategies first. There are quite a few aspects that Starbucks could improve regarding their mobile marketing efforts. In particular, it needs to provide customers with information on the average waiting time for order preparation and facilitate the payment process using Apple Pay (Bidgoli 193). Also, at the moment, the mobile application does not provide information about the staff of a particular coffee shop. However, most importantly, the enterprise could minimize the need to purchase a physical loyalty card in order to initiate the payment process through the app. Concluding Points Thus, it can be concluded that the mobile marketing strategy employed by Starbucks turned out to be effective since it allowed attracting new customers while also retaining loyal clients through improved user experience. Other companies could consider introducing a built-in payment system and virtual reality feature as client-side solutions. Nevertheless, Starbucks also has issues that require improvement, which are related to order delivery and payment via the app. Get your 100% original paper on any topic done in as little as 3 hours Learn More Works Cited Bidgoli, Hossein. MIS: Management Information Systems. 8th ed., Cengage Learning, 2017. Richter, Felix. “Starbucks Brings Mobile Payment to the Masses.” Statista, 2018. Web.
There has been an argument as to whether whistle blowing should be considered an essential aspect when it comes to the issue of enhancing a better business continuity mismanagement system. This is the reason why the US Labor Department is collecting comments that will give a good direction and come up with new regulations that will have a long term effect of protecting whistle blowers (Honour 1). Whistle blowers are mainly workers who voice issues about security, safety, and health concerns. Business continuity is an activity performed by many organizations to ensure that critical business functions are accessed and guaranteed by all the stakeholders concerned. Many people have assumed that business continuity should only be conducted or done when an organization is faced by a disaster (Honour 2). Although this should not be the case, however, business continuity should be done or performed daily to maintain recoverability, service, and consistency. It has been argued that encouraging employees to report risky business operations in their organizations supports and enhances business continuity. This is because silenced workers will never feel free to work well. In the long run, their potential will not be realized. This is a very important issue when looked at from a business continuity perspective. Perhaps there is a need to ponder on the question of whether or not whistle blowing should be encouraged as an essential aspect of business continuity (Honour 3) about disaster recovery and emergency management. Workers should be given a voice as a way of getting a better insight into a given organization. This way, they stand a chance to better understand the risks faced by an organization without the knowledge of others or better still the public at large. These risks can be hidden, but they may have a very large business impact on a given organizations’ continuity. For efficient business continuity, there is a need to have good disaster management and recovery measures that will ensure that huge losses are not incurred. Without good measures to mitigate such eventualities, an organization will find it hard to operate well, and this may come up as a challenge in a bid to compete well. As much as whistle blowing can be encouraged as a way of identifying disasters in advance, several boards have seen this as a threat to their organizational and business operations and hence, not a good thing (Honour 2). Instead of appreciating such employees, the management and boards have seen them as enemies who are reporting the organization to a given regulatory or government body that will eventually punish them. Get your 100% original paper on any topic done in as little as 3 hours Learn More This should instead be seen as a way of encouraging employees to voice their concerns on areas they think the organization is not doing well. Organizations are advised to have their whistle blowing structures internally as a way of assessing their performance. If this is done efficiently, employees will feel that they are highly valued listened to and empowered (Honour 3). This will give them another reason to work harder and ensure that the organization is successful. In other words, they will feel that they are part of the organization and in the process, give it their best. This approach will likely create a good business continuity and disaster management approach. There are occasions where employees might see something that will hurt the organization and keep it to themselves. It might be because they fear to talk about such issues due to the consequences that might befall them (Honour 4). Business continuity will only be termed effective if the latest information that preceded it is well catered for. Managers know very well that business continuity will only be guaranteed if there is a proper business impact analysis (Honour 4). There is no way that an organization can achieve considerable success without a proper business impact analysis. The impact analysis should be done while looking at the possible negative and positive scenarios that might befall an organization as a result of a given action. This process will be flawed if employees and the management don’t work together harmoniously as expected (Honour 4). They should not feel that they are working under pressure from others. In this case, they should be free to reveal whatever they feel is important towards positive continuity of the organization. This means that they should be in a better position to disclose processes and procedures that are risky or disastrous than they may be presumed to be. From this explanation, it is obvious that whistle bowing can come in handy in enhancing the achievement of proper disaster management and business continuity. A good whistle blowing process will ensure that there is a lot of confidentiality in information delivery, which will make it easy for participants to input their concerns without any fear (Honour 5). Whistle blowing will make it easy to identify new disasters that have not been factored in a given organizations’ business continuity plan. If employees are encouraged to whistle blow, an organization will be safe from any disasters that might pose a threat to its continuity (Honour 6). We will write a custom Article on Disaster Recovery and Emergency Management – Business Continuity specifically for you! Get your first paper with 15% OFF Learn More Works Cited Honour, D. Why encouraging employees to report risky processes and behaviors supports business continuity. 2010. Web.

RSCH 8210 WU Week 11 Testing for Bivariate Categorical Analysis Research Paper

RSCH 8210 WU Week 11 Testing for Bivariate Categorical Analysis Research Paper.

Testing for Bivariate Categorical AnalysisTo prepare for this Assignment:Review Chapters 10 and 11 of the Frankfort-Nachmias & Leon-Guerrero course text and the media program found in this week’s Learning Resources related to bivariate categorical tests.Using the SPSS software, open the Afrobarometer dataset found in this week’s Learning Resources.Next, review the Chi Square Scenarios found in this week’s Learning Resources and consider each research scenario for this Assignment.Based on the dataset you chose and for each research scenario provided, using the SPSS software, choose a categorical data analysis and run a sample test.Once you perform your categorical data analysis, review Chapter 11 of the Wagner text to understand how to copy and paste your output into your Word document.For this Assignment:Write a 1- to 2-paragraph analysis of your categorical data results for each research scenario. If you are using the Afrobarometer Dataset, report the mean of Q1 (Age). In your analysis, display the data for the output. Based on your results, provide an explanation of what the implications of social change might be.Use proper APA format, citations, and referencing for your analysis, research question, and display of output.By Day 7Submit your Assignment: Testing for Bivariate Categorical Analysis.
RSCH 8210 WU Week 11 Testing for Bivariate Categorical Analysis Research Paper

Economic Growth as an Indicator for Development

essay writing help “Economic Growth is a necessary but not sufficient condition of Economic Development”. Comment on the limitations of economic growth and the importance of accounting for multiple dimensions of well-being beyond growth. You can use empirical findings or a case study to support your arguments. Introduction Economic growth is the percentage rate of increase in the monetary value of all the final Goods and Services produced in a period of time. Economists usually measure economic growth in terms of Gross Domestic Product (GDP) or Gross National Income(GNI) Economic development is the improvement in the well-being of the people of a nation in its economic, political and social spheres. The economic growth is an important condition in the economic development of a country. But the economic development is a much broader connotation than just the economic growth. The economic development of the people cannot be measured only by the increase in their income. The other measures of economic development include the social conditions like literacy rate, life expectancy and various other indicators which will influence the social and cultural well-being of a human being. In trying to explain the necessity but the insufficiency of economic growth on economic development, this essay will first discuss what economic development actually means and the different dimensions it entails. Then the essay will go on to talk about the importance of economic growth in the framework of economic development and yet why it falls short of being sufficient enough for economic development. The last section will include a case study of India, which has shown remarkable economic growth post liberalisation and still lags behind in economic development indicators reinforcing the limitations of economic growth. Economic Development over the years The term ‘Development’ has its origins immediately after the second world war, when the United States initiated a massive reconstruction programme. From then on, the word has been researched and analysed and has over the years taken various different forms and meanings. In strictly economic terms, development has traditionally meant achieving sustained rates of growth of income per capita to enable a nation to expand its output at a rate faster than the growth rate of its population (Todaro.P 2010: 14). Till the 1970s it was believed that an increase in the gross national income(GNI) will trickle down to the masses in some form and help improve their social and economic indicators. But very soon it was realized that the even with the increase in the national income the social indicators of many underdeveloped countries remained stagnant. So the term economic development was redefined in terms of the reduction in poverty, inequality and unemployment within the context of the growing economy (Todaro.P 2010: 15) The decade before the turn of the millennium saw many new changes in the way economic development was being understood. The goals of economic development became much wider by including improvements in the income distribution, environment, health and education (Pheng L., Dang G.,2015) During this period Sen’s work brought about the broadest perspective of development goals (Pheng L., Dang G., 2015: 13) As Sen put it, “Economic growth cannot be sensibly treated as an end in itself. Development has to be more concerned with enhancing the lives we lead and the freedoms we enjoy.” (Todaro.P 2010: 16). This marked a complete shift in the way economic development was perceived and appraised. Income or wealth which were till then seen as ends in itself began to be seen as means to achieving other purposes. The focus of economic development shifted towards the quality of life that individuals are able to achieve ( Dang. 2014: 461). The idea is that the quality of life cannot be determined or evaluated only by the resources an individual has but the ability of the individual to use the resource(capabilities), the reality of using the resource (functioning’s) and the psychic state of the individual in using the resources (Utilities) (Sen.A, 2000). The development economists started to focus on health and education after Sen’s capability approach since to convert the characteristics of commodities(resources) in to functionings, and to have the freedom of the choice of functionings, in most important cases, requires good health and education. (Todaro.P, 2010 : 18). Thus to broadly conceptualize economic development, it basically means improvement in the well-being of an entire society and its social systems (Todaro.P, 2010) Multiple dimensions of well-being From the previous section it was clear that the wellbeing is inherently multidimensional (Decanq. A., Lugo M, 2013: 8). It should take in to account the objective condition of people and their subjective assessments of their lives. (Adler.A 2016). The objective dimension of the well being largely originates from Amartya Sen’s work where he talks about the capabilities individuals should have to live fulfilling lives. (Western M, Tomaszewski W 2016). Some core human capabilities include bodily health, bodily integrity, the ability to use the senses to think and to imagine, the ability to express emotions, to exercise practical reasons and autonomy with respect to one’s own life, to affiliate, to live with dignity etc (Nussbaum M 2003). The subjective dimension of wellbeing. Broadly the subjective dimension includes the freedom defined in terms of what an individual values doing and being. ( Sarah c, Mcgregor A,White C., 2018). The freedom to doing and being will be contingent on other components of well-being like education and in turn becomes a pre- condition for a few constituents of well-being like equity and fairness.( Millennium Ecosystem Assessment, 2005). There is also a relational dimension which should be considered, the social relationships of an individual, where the well-being of the individual is seen as a social or collective, going beyond the individual (ibid). Mankind’s activities have adversely affected the functioning of the earth that the wellbeing of an individual is also based on the quality of the ecosystem (Helne T, Hirvilammi T,2015). This adds an environmental dimension to the well-being conundrum. If we are to achieve the development in our societies then it is necessary to reform our statistical data collection from being focussed on measuring progress in terms of production and consumption, to measuring in terms of human wellbeing ( Sarah c, Mcgregor A,White C., 2018) But it is very important to understand the various dimensions that influences the well-being of individuals before it can be operationalized into a single index of well-being. Many studies have been made to operationalize all these dimensions of well-being in to a single index. Indices like Human development index(HDI), Multidimensional poverty index(MPI) have been created to measure progress and well-being of a society. Studying the indices is beyond the scope of this essay. Economic growth Foremost, it is important to understand the term economic growth in it fullness. Economic growth is an increase in the capacity of an economy to produce goods and services, compared from one period of time to another (Investopedia 2005). Often, but not necessarily, aggregate gains in productivity correlate with increased average marginal productivity(ibid) So in simple terms, more the economy grows, more the income of the individuals (Income per capita) of the nation whose economy has grown. More income naturally leads to increased consumption, investment and savings. Relationship between economic growth and economic development The presumption for many years was that increased income(Economic growth) of a nation will lead to economic development. It was Richard Easterlin who first questioned the idea that GDP will lead to economic development. For the last few decades the debate on whether higher GDP will lead to greater social prosperity has existed. (Adler A, Seligman M, 2016:2) So what exactly is the relationship between economic growth and economic development? Before the start of industrialization, the average real life standards of the rich countries were no more than three times as great as the other poorer countries of the world (Todaro P, 2010: 78). But in the previous century the today’s developed countries have enjoyed far higher incomes than the developing (Poorer) countries. (Pritchett L, 2010: 4). The living standards of these developed countries have also increased many folds in the last century compared to the developing countries. I(ibid). Clearly there exists a strong relation between economic growth and human development. The economic growth provides the resources for human development and without these resources it will not be possible to achieve human development which is clear from the average real life standards of the rich countries today when compared to the poorer countries. (Ranis G, Stewart F, Ramirez A, 2000: 197). A study done on 14 countries from the 1990s by the UK department of International development, the world bank, shows that poverty fell in the 11 out of the 14 countries which experienced economic growth and poverty grew in the other 3 countries where the economy showed negative growth ( World bank 2005). ) Reduction in poverty will surely aid in increase in Human developmentThe study of Gallup world poll data by (Deaton A, 2008) shows that “Each doubling of GDP is associated with a constant increase in life satisfaction” ( Easterlin A, 2013) . Thus it is clear the economic growth in GDP per capita is a very important and a necessary condition for economic development. But the rankings done on the basis of HDI for 37 countries in 1993 did not match with the income rankings. (UNDP 1996 :5) Some countries like Colombia has achieved high human development though their income per capita is quiet modest, while countries like South Africa and Gabon having more than twice and thrice the per capita incomes of Colombia shows lower human development. (ibid). Social Progress Index (SPI) is one of the new measures developed to evaluate human development. The SPI takes in to account 53 social and environmental indicators under three headings : basic needs, the foundation of a well being and opportunity. (Economist 2016) When SPI is shown against the economic growth (GDP Per capita). The curve is not linear showing that the social progress slows down after a lever of GDP per capita is reached. Thus there is diminishing returns for GDP when a threshold is reached. Figure 1 : Source : Social progress indicative, The economist(June 2016) The OECD( Operation for Economic Cooperation and Development, 2013) devised an index for the measurement of economic development called as the better life index. They chose 11 domains to be included in the index and ranked all the 11 domains individually as well as had a composite index based rank. Different ranks emerged when the domains are taken differently. If only life satisfaction is taken countries like Switzerland, Norway tops the list. But the wealthiest country is the United States. Australia which tops the list in the composite index does not even come in the top ten of the highest income generating countries and the US comes a distant 14th in life satisfaction and 6th when all the 11 domains are taken together. (Alder A, Seligman M, 2016). As the many studies clearly suggests GDP as the single measure of economic development raises many questions. There are many facets of economic development which cannot be quantified like the relational dimension, the environmental factors, the psychological factors etc (Adler A, Seligman M, 2016). Other issues like Justice, governance, equity and fairness which plays vitally important roles in the well-being of the humans could never improve with increase in GDP. Finally, economic growth is not an end in itself, while economic development or the well-being of the individual is a means to an end. Of course economic growth is necessary for economic development, as all indicators show that the richer countries have better well-being indices. But after a point there are far too many factors which influence the economic development of a country or the well-being of an individual to reduce it to an index as small as economic growth. Economic growth is not sufficient enough to represent economic development of a country. Conclusion Economic growth is a very important indicator in the development of a country. More the income, more the capital spent on welfare mechanisms and social structures. But income in itself is not an accurate measurement of the development story of a country. Income distribution is equally or sometimes more important. Thus GDP per capita is a better measurement of the economic growth than GDP in itself. But Increased GDP per capita is just a means to an end, higher economic development of a country or increased well-being of individuals. Thus Economic growth is a necessary but not a sufficient condition of economic development Many countries have started using different indicators to measure the economic development of their countries. Bhutan is using Gross National Happiness (GNH) to design its policies. “France has recommended that the statistical offices of the world should incorporate questions to capture people’s life evaluations, hedonic experiences, and priorities in their own terms” (Adler A, Seligman M, 2016 : 17). The United Kingdom has released their own Gross Domestic Well-being. The OECD had developed its own Better life index for its countries. a United Nations resolution encouraged Member States “to pursue the elaboration of additional measures that better capture the importance of the pursuit of happiness and wellbeing in development with a view to guiding their public policies” (UN General Assembly Resolution A/65/L.86). In the broader context, these indices help the governments of the various countries and the international organizations to formulate policies which will improve the quality of life of the masses. Bibliography 1.2 Economic growth and development [WWW Document], n.d. URL (accessed 10.30.18). 07e0c52e-39c2-4e09-a9ac-cc8ac99071c6.pdf, n.d. 328850Pro1poor1growth1in1the11990s.pdf, n.d. 40700982.pdf, n.d. Adler, A., Seligman, M.E.P., 2016. Using wellbeing for public policy: Theory, measurement, and recommendations. International Journal of Wellbeing 6, 1–35. Bank, T.W., 2005. Pro-Poor growth in the 1990s : Lessons and insights from 14 countries (No. 32885). The World Bank. Barro, R.J., 1996. Determinants of Economic Growth: A Cross-Country Empirical Study (Working Paper No. 5698). National Bureau of Economic Research. Barro, R.J., 1991. Economic Growth in a Cross Section of Countries. Q J Econ 106, 407–443. Benjamin, D.J., Kimball, M.S., Heffetz, O., Szembrot, N., 2014. Beyond Happiness and Satisfaction: Toward Well-Being Indices Based on Stated Preference. Am Econ Rev 104, 2698–2735. BLI2013-Country-Notes.pdf, n.d. Coulthard, S., McGregor, J.A., White, C., 2018. Multiple dimensions of wellbeing in practice, in: Schreckenberg, K., Mace, G., Poudyal, M. (Eds.), Ecosystem Services and Poverty Alleviation. Routledge, pp. 243–256. Coulthard, S., McGregor, J.A., White, C., n.d. Multiple dimensions of wellbeing in practice 14. Dang, A.-T., 2014. Amartya Sen’s Capability Approach: A Framework for Well-Being Evaluation and Policy Analysis? Review of Social Economy 72, 460–484. Dang, G., Sui Pheng, L., 2015. Theories of Economic Development, in: Dang, G., Sui Pheng, L. (Eds.), Infrastructure Investments in Developing Economies: The Case of Vietnam. Springer Singapore, Singapore, pp. 11–26. Decancq, K., Lugo, M.A., 2013. Weights in Multidimensional Indices of Wellbeing: An Overview. Econometric Reviews 32, 7–34. Economic development, 2018. . Wikipedia. Gross Domestic Product (GDP) vs Gross National Product (GNP) – Difference and Comparison | Diffen [WWW Document], n.d. URL (accessed 10.31.18). Gu, S., n.d. A World History of Economic Development: A Contribution to the Theory of Economics. Haidt, J., 2006. 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Expression of Recombinant Green Fluorescent Protein (rGFP)

Expression and Purification of recombinant Green Fluorescent Protein (rGFP) from E. coli using Ni2 -Agarose Column Chromatography. Andrea Bustamante Janakikeerthika Darmarpandi Abstract Green Fluorescent Proteins are vital components of bioluminescence in marine animals. There unique ability to withstand and recover from harsh conditions and regain fluorescence was of great interest. The purpose of the following set of experiments was to express and purify a His6-Xpress epitope tagged recombinant form of Green Fluorescent Protein grown and harvested from E. coli. The desired protein is initially released into solution using the properties of freeze-quick thaw cycles that then help release the contents of the nucleus of neighboring bacteria following a chain reaction. It is then submitted through a Ni2 -agarose affinity chromatography column where the target protein was purified. The resulting wash and elution fractions where run through a Bradford assay, SDS-PAGE/Coomassie blue staining, and a Western blot to determine the molecular weight of the protein to be 32kDa. The overall specific activity was determined to be 433000 RFU/ mg of total protein with a resulting 20 percent purity. The results show that expression and purification of rGFP from bacterial cells was possible. Introduction Aequorea victoria is a jellyfish capable of producing a green fluorescent light when Ca2 ions activate a photoprotein, known as aequorin, which excites Green Fluorescent Protein (GFP). Wild type GFP is a 27kDa, homodimer composed of 238 amino acid residues that absorbs light at an excitation wavelength of 395nm (blue light) and emits light at an emission wavelength of 510nm (green light). Aequorea victoria GFP has a distinctive three dimensional structure that encases a chromophore (formed by cyclization of Ser65-dehydrogenized Tyr-Gly67) and allows for stability under harsh conditions (Prasher, 229-230.) . This structure allows for regaining of fluorescence even after the protein has been denatured upon removal of the denaturant. Therefore, GFP’s are extremely stable to changes in pH, temperature, oxidation and reduction, and chemical reagents (Pan, Pickett, and Rippel 225.) Poly-histidine tags involve addition of a series of histidine residues to the N or C terminus of a protein of interest. Poly-histidine tags are affinity tags that serve to facilitate protein purification by exploiting the positively charged histidine residue’s affinity for negatively charged columns. This series of experiments involved a six repeat histidine codon contained within a DNA plasmid which resulted in a recombinant Green Fluorescent Protein that contained a six residue histidine tag located at the N-terminus. The His­6 tagged recombinant Green Fluorescent Protein was then subjected to Ni2 -agarose column affinity chromatography. Ni2 -agarose affinity chromatography allows for the purification of poly-histidine tagged proteins due to the selectivity and affinity of the Ni2 -agarose matrix for His6 tagged proteins. rGFP binds the column due to the interactions between the His6 tagged proteins in the mobile phase with the metal Ni2 ions immobilized within the matrix in the stationary phase. The Ni2 ions contained within the matrix are capable of binding electron rich molecules including histidine residues and allowing most other molecules to pass unbound. This results in the binding of the desired protein to the column and the purging of most undesired proteins and contaminants from the column into wash fractions (Ninfa, et al. 100-101.) The column was then subjected to imidazole, which competes with rGFP for Ni2 ion attachment, and this allows for the elution of the target protein. Due to its unique properties, isolation of GFP was of great interest and expression and purification were the main focus of the following series of experiments. A suitable way to accomplish this was devised using the combination of poly-histidine tagging and affinity chromatography. The purpose of this experiment was to express and purify a six-Histidine tagged recombinant form of Green Fluorescent Protein from E. coli through the use of Ni2 -agarose affinity chromatography. After expression and purification, a Bradford assay was performed to estimate total protein amount. This was followed by SDS-PAGE/Coomassie blue staining to determine purity and molecular weight. The confirmation of the presence of rGFP was done using the Western Blot. Materials and Methods Growth of G strain In a test tube, 10ml of liquid LB growth media containing 100ug/ml Amp and 25ug/ml Cam was inoculated with a single bacterial colony of strain G (BL21(DE3)uv>) and was allowed to grow overnight at 37°C. The culture was shaken until saturated. In a flask, 500ml of liquid LB media (pre-warmed to 30°C) was inoculated with about 4 ml of the saturated overnight culture (or until the 500ml culture reached an OD600 reading of 0.1) and allowed to grow at 37°C until the OD600 reading reached 0.5. At approximately OD600 ~0.5, or time zero, 1ml of the culture was harvested into a 1.5ml centrifuge tube and pelleted. The supernatant was discarded and the “G0” pellet stored at -20°C for later use. The remaining culture was induced with 1mM IPTG and allowed to grow. After 3 hours, 1ml of the culture was harvested into a 1.5ml centrifuge tube and pelleted. The supernatant was discarded and the “G3” pellet stored at -20°C for later use. An additional 15ml of the IPTG induced culture was harvested into a 15ml centrifuge tube and pelleted. The supernatant was discarded and the “G3-15ml” was stored at -20°C. Preparation of rGFP Crude Extract Immediately after removal of the “G3-15ml” pellet from freezer, breaking buffer [10mM Tris, pH 8.0; 150mM NaCl] was added into the centrifuge tube. The breaking buffer was pipetted up and down (being careful not to introduce air) until pellet had thawed and homogeneity was reached. The solution was transferred into a 1.5ml centrifuge tube, vortexed for 5 minutes, labeled and placed in 37°C water bath for 10minutes after which the centrifuge tube was transferred to a rotating platform shaker in a dry air 37°C incubator for 20 minutes. After lysis, the mixture was centrifuged at 14000xg, 4°C, for 10 minutes. In a dark room in the presence of a hand held UV light, the fluorescence of the pellet and supernatant where observed the recorded. The supernatant was then decanted and care was taken not to get the pellet back into the supernatant as centrifugation would be required if this did occur. This supernatant was the GCE (rGFP crude extract) Preparation of Ni2 -agarose Column In a 3ml plastic syringe, enough glass wool was placed into the well to cover up to the 1/4 ml marking. The syringe was secured onto a ring stand and placed perpendicular to the ground. About 100ul of breaking buffer was pipetted into the top of a closed luer-lock and allowed to overflow. 1ml of buffer was then pipetted into the syringe column and the luer-lock was immediately screwed onto the syringe. An additional 2ml of breaking buffer was added to the column and several drops of buffer were allowed to flow out. The luer-lock was then returned to the closed position. A total of 500ul of breaking buffer was added to the column and then 1ml of a 0.5ml bed volume Ni2 -agarose slurry was added to the column. The luer-lock was opened and agarose matrix was allowed to “gravity pack.” The column was pre-equilibrated with 5ml of breaking buffer and then the luer-lock was returned to the closed position. Ni2 -NTA Chromatography Separation Procedures 100ul of GCE was transferred into a centrifuge tube, labeled, and set aside. Breaking buffer was added to remaining GCE if content was less than 1ml. GCE was slowly applied to the Ni2 -agarose column and allowed about 5-10 minutes for protein to bind to column. The luer-lock was opened and 0.5ml of effluent was collected into 1.5ml centrifuge tube and labeled W1. This was repeated with the subsequent effluent labeled W2.The column was then observed under an ultraviolet light and fluorescence recorded. The column was then washed with 4ml of buffer in 0.5ml increments. The effluent was collected and labeled W3 to W10. The column was then washed again with a total of 5ml of breaking buffer. This effluent was discarded. A total of 5ml of elution buffer containing 10mM Tris, pH 8.0; 150mM NaCl, 300mM imidazole was added to the column in 0.5ml increments. The eluents were collected and labeled E1-E10.The column was then observed under a UV light and the fluorescence recorded. The W1-W6 and E1-E6 fractions were also observed under UV light and their fluorescence recorded qualitatively. Determining Total Protein Amount A standard curve was created using six different samples of Bovine Serum Albumin (1mg/ml) of known amount. The amounts of BSA used all had a final volume of 50ul and included 0ug, 3ug, 5ug, 10ug, and 20ug total proteins. A total of 1ml of Bradford reagent was added to each, vortexed, and allowed to incubate for 10 minutes. The results where read using 200ul in a microtiter dish and read using a microplate reader set to 595nm. The results where plotted on a graph as absorbance (595nm) vs. BSA (ug) and a best fit line was drawn. The Bradford assay was then performed once on the W1-W6 and E1-E6 samples. Any samples whose absorbance fell outside the standard curve were repeated less sample in the assay. Once all samples fell within the standard curve, the Bradford assay was repeated two more times for each sample. The total protein amount was then extrapolated from the standard curve using the absorbance values. Estimating Purity and Molecular Weight The SDS-PAGE was prepared using a 12 percent resolving gel that was poured between the Bio-Rad glass plate “sandwich” and allowed to polymerize. A 5 percent stacking gel was prepared and added on top of the resolving gel, a comb was inserted, and the gel was allowed to polymerize. Once that polymerized, the combs were removed and the electrophoresis tank was set up. 15ul of G0, G3, GCE, W3, W4, E2, and E3 samples were added to the SDS-PAGE along with a standard molecular weight ladder. The samples were electrophoresed at 200volts for 45 minutes. The gel was then stained using Coomassie blue dye and the stain removed. Confirmation of rGFP 2-β-mercaptoethanol was added to the centrifuge tubes containing the G0, G3, GCE, W3, W4, E2, and E3 samples and were loaded along with a molecular weight ladder and electrophoresed as described above. The stacker was removed and the resulting gel set up for transfer onto a nitrocellulose membrane for Western Blot analysis. The overall setup required a “building up” of components with the positive electrode base on the bottom, followed by filter paper soaked in transfer buffer, nitrocellulose paper above that, the SDS/PAGE layer, another layer of filter paper soaked in transfer buffer, Western blot solution was poured over all the components, and finally the negative electrode lid was locked into position. To ensure transfer, the nitrocellulose gel was stained using Ponceau S and allowed to incubate for two minutes on a rocker and then destained using ddH2O. The membrane was then blocked using 5% non-fat dry milk/TBS solution and incubated for 30 minutes on a rocking platform. This was then and washed three times with 0.05%Tween 20/TBS with 5 minutes of incubation between each wash. It was then probed with mouse IgG anti-Xpress epitope MAb solution and allowed to incubate for 45 minutes. The 0.05%Tween 20/TBS wash was repeated in triplicate. A secondary probe using sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution was performed as above and then washed in triplicate. The nitrocellulose gel was developed using TMB until desired intensity was reached and development was stopped with water and results recorded immediately. Results The expression of the target protein was doubly repressed in the G0 (uninduced) sample of E. coli. First, the Lac repressor protein binds to the lac operator and prevents transcription by T7 RNA polymerase (Garrett and Grisham 915-916). Second, T7 RNA was repressed by lysozyme protein that binds to T7 RNA polymerase and inhibits transcription. Expression of rGFP in the G3 (3 hour post induction) sample was made possible through the use of IPTG (Garrett and Grisham 914.) The purpose of IPTG was to repress the Lac repressor which resulted in T7 RNA polymerase being able to transcribe DNA downstream of the T7 promoter and expression of His6-Xpress-GFPuv, resulting in the fluorescent capable recombinant Green Fluorescent Protein. (Figure 1) This resulting recombinant GFP is a 279 amino acid protein. rGFP has a six Histidine tag at its N terminus between amino acids 5 and 10, an Xpress epitope between amino acids 24 and 31, Green Fluorescent Protein between amino acids 39 and 277, and a 3 amino acid end tag between amino acids 277 and 279. The chromophore is found between amino acids 103 and 105 in the DNA sequence. (Figure 2) Results of Ni2 -agarose affinity chromatography and Bradford assay indicated that the E3 (elution 3) sample contained the most rGFP activity with approximately 18,600 RFU (relative fluorescent units) and an estimate 43ug of total protein. The specific activity calculated for the sample was 433000 RFU/ mg of total protein. (Figure 3) The SDS-PAGE/Coomassie staining gave an estimate molecular weight for rGFP of 32kDa based on a total traveled distance of 2.3cm along the SDS/PAGE. The overall purity of the band was approximately 20 percent. The higher molecular weight band was most likely contaminants at about 45kDa and the lower molecular weight band was possibly a result of the degradation of the c-terminus at 27kDa. (Figure 4) Western Blot indicated prominent bands in the E3, E2, GCE, and G3 lanes. Lanes W4 and W3 showed very light bands and lane G0 shows an absence of bands. All visible bands appear at about 32 kDa and therefore confirm the presence of rGFP. (Figure 5) Conclusion The successful expression and purification of recombinant Green Fluorescent Protein is significant in the scientific community due to the possible uses for it in the future. Green Fluorescent Protein is significant because it provides an inexpensive and relatively easy method of detection. The possibility for real time detection means result could be obtained in real time. Future experiments will focus on linking rGFP to proteins during transcription and translation. This would result in a desired protein with a GFP tag whose fluorescence can then be used for identification. This should result in the ability to locate a target protein using the fluorescence of rGFP. Future applications of GFP could include incorporation into the genetic code of small mammals. These could encode fluorescent neurons which in turn could help further research in areas such as nerve tissue regeneration or other advances in neurobiology. Its unique properties of endurance could be exploited to understand how it can endure harsh environments and still regain functionality after remediation. This would have significant applications in molecular and cellular biology in understanding cellular degeneration and how help patients with diseases involving cellular degeneration. Bibliography Pan, Jing, Elizabeth Pickett, and Scott Rippel. Biochemistry Laboratory Lecture Notes. Dallas: UTD copy center, 2013. 225-289. Print. Pan, Jing, Elizabeth Pickett, and Scott Rippel. Biochemistry Laboratory Manual. Dallas: UTD copy center, 2013. 38-77. Print. Prasher, Douglas C., Virginia K. Eckenrode, et al. “Primary Structure of the Aequorea victoria green-fluorescent protein.” Gene. 111. (1992): 229-233. Print. Garrett, R., and Charles M. Grisham. Biochemistry. 4th ed. Belmont, CA: Brooks/Cole, Cengage Learning, 2010. Print. Ninfa, Alexander J., and David P. Ballou. Fundamental laboratory approaches for biochemistry and biotechnology. Bethesda, Md.: Fitzgerald Science Press, 1998. 89-107. Print.

Lesson Plan Review: Effects of the War Report (Assessment)

Lesson Plan Review: Effects of the War Report (Assessment). The lesson is devoted to the effects of the American Civil War. During the lesson, students will learn about 13th, 14th, and 15th Amendments and discuss their significance. Students will also learn about Booth’s plan concerning Lincoln’s kidnapping and Booth’s reasons for Lincoln’s assassination. Students will learn about reconstruction, as well. The lesson plan is very detailed, and the lesson is quite effective. Thus, the strengths of the lesson plan are as follows. The teacher uses a PowerPoint presentation, which can help students grasp the information. It also enhances students’ attention. The teacher has several hand-outs. The activities described are engaging and creative as students do not simply learn about some facts, dates, and names. They discuss the events and try to see them in the context of the USA of the 19th century. Hence, students learn to think critically. The lesson plan also includes assessment section. Thus, it will be possible to understand whether the lesson is effective and whether students have grasped the necessary information. Nonetheless, the lesson plan has one downside. When discussing the Constitution and Booth’s plans, the teacher uses certain hand-outs and tells about those events. However, it could be more effective and engaging to have a short video clip to make students feel the atmosphere of the USA of that period. Thus, to improve the lesson, the teacher should find a video or several video clips to make the lesson more expressive. This can be a part of a documentary or even a scene from a drama. There are numerous documentaries on the Civil War and Lincoln. Such movies as The Day Lincoln Was Shot (1998), Gone with the Wind (1939), North and South (2004) can be used. The use of video could help students picture the atmosphere in the United States. Lesson Plan Review: Effects of the War Report (Assessment)